UP-REGULATION OF GAP JUNCTIONAL INTERCELLULAR COMMUNICATION IN IMMORTALIZED GONADOTROPIN-RELEASING-HORMONE NEURONS BY STIMULATION OF THE CYCLIC-AMP PATHWAY

Increased gap junctional intercellular communication induced by agents that stimulate the adenylyl cyclase/cAMP pathway was observed in the GnRH-secreting neuronal cell line, GT1-7, and possible underlying mech anisms were examined. A 24-hour treatment of GT1-7 neurons with 100 mu M dibutyryl cAMP + 100 mu M IBMX or with 2 mu M forskolin increased b y greater than 2-fold the percentage of cells that were dye coupled, u sing the noninvasive dye coupling assay, fluorescent recovery after ph otobleaching (FRAP). Longer treatment times (48 h) and higher concentr ations of dibutyryl cAMP (500 mu M) did not further increase the perce ntage of dye-coupled cells, while there was no increase in dye couplin g observed between untreated cells and cells treated for 2 h or less. The increase in dye coupling induced by dibutyryl cAMP/IBMX was inhibi ted by octanol or dieldrin, agents known to block gap junction-mediate d intercellular coupling in other cell types. Western blot analysis of total protein or membrane protein-enriched extracts revealed no appar ent difference in the cellular levels of connexin 26, a connexin subty pe previously shown to be expressed by GT1-7 cells, between untreated cells and cells treated for 24 h with dibutyryl cAMP/IBMX or forskolin . In addition, expression of connexin 32 or 43 protein before or after treatment was not detected. On the other hand, a dramatic increase in both the number of neurites and neurites that immunostained positive for connexin 26 was observed in dibutyryl cAMP/IBMX-treated cells. We hypothesize that the observed increase in dye coupling between GT1-7 n eurons following stimulation of the adenylyl cyclase/cAMP pathway resu lts from an augmentation of cell-cell contacts due to an increased num ber of neurites containing gap junctional plaques, possibly through an effect on cellular differentiation.